The DNA binding landscape of grapevine NAC transcription factors

Transcription factors (TFs) are DNA-binding proteins that regulate gene expression by recognizing short motifs found in the promoter region and at other sites upstream and downstream of the transcriptional start site. By linking genes into regulatory networks and hierarchies, plant TFs help researchers to understand the key mechanisms of gene regulation in crops, and accordingly they will play an increasingly important role in agriculture. Approximately 10% of plant genes encode TFs, many of which are known for their roles in plant growth, development and stress tolerance. The activity of TFs is influenced not only by the DNA sequence motifs they recognize, but also by epigenetic mechanisms, cofactors and cooperation with other TFs in molecular complexes. The work described in this dissertation focuses on the NAC family of TFs in grapevine. The NAC family is named after its three founding members: NO APICAL MERISTEM (NAM), Arabidopsis thaliana transcription activator factor 1/2 (ATAF1/2) and CUP-SHAPED COTYLEDON 2 (CUC2). Grapevine NAC proteins (VvNACs) regulate growth, development, stress responses and defence mechanisms, including the control of berry ripening and leaf senescence. The work herein is part of a project of national interest (PRIN) that sees the collaboration of the universities of Udine, Padua and Rome. The aim of the project was to study the regulation of gene expression in grapevine and to analyse the genetic and epigenetic determinants. The focus of the work set out in the following chapters was to analyse the VvNAC family by DNA affinity purification sequencing (DAP-Seq). This is a rapid and inexpensive approach for the identification of TF binding sites, which involves the cloning of TF genes, in vitro expression, the capture of target sequences from a genomic DNA library, the affinity purification of TF–DNA complexes, and the analysis of the captured DNA by next-generation sequencing to generate cistrome and epicistrome maps. Analysis of the grapevine cistrome/epicistrome is important due to the scientific and economic value of this crop. Understanding how growth, development and stress responses are regulated will provide insight into the development of high-quality berries, which are required to meet the quality standards to produce fruit, juices and wines. It is also scientifically intriguing because the grapevine has a complex 487-Mb genome with a high content of transposable elements, making it a useful model in which to study how gene expression is regulated in the context of genome evolution DAP-Seq was used to sketch the cistrome for all 74 known VvNAC proteins. Large DNA-Seq datasets were generated using DNA isolated from young grapevine leaves of the cultivar Cabernet Franc and were then refined by statistical and bioinformatic analysis. In this manner, we validated the results for 24 NACs, which were studied in more detail to determine their biological functions. This work suggests that VvNAC02 regulates plant growth, VvNAC04 regulates biotic and abiotic stress responses, VvNAC05 has a role in berry development, VvNAC16 regulates flower development and inflorescences, VvNAC22 induces defence responses against pathogens, VvNAC26 controls berry development and maturation, VvNAC34 regulates leaf senescence, VvNAC40 facilitates the response to water stress, and VvNAC71 has a more general role in biotic and abiotic stress responses.DAP-Seq is an in vitro technique, so it does not account for in vivo factors that affect TF activity, such as DNA/histone modification and chromatin structure. We therefore compared the DAP-Seq data to datasets gathered using two in vivo techniques: the dual luciferase reporter assay (DLRA) and chromatin immunoprecipitation sequencing (ChIP-Seq). We tested several variations of the grapevine ChIP-Seq protocol described by Foresti (2021) and achieved significant improvements by identifying the importance of a pre-treatment washing step to eliminate contaminating plant pathogens, the use of fresh rather than frozen material for immediate fixation, and the advantage of a double fixation step using disuccinimidyl glutarate (DSG) and formaldehyde. We identified several VvNAC60 targets in agreement with previous studies, such as Senescence-associated protein (VIT_02s0012g00280), which is involved in leaf senescence. This work describes one of the most important families of TFs in grapevine and provides new information showing how these NAC proteins interact in networks to activate target effector genes involved in diverse pathways that control plant growth, development, stress responses and defence. A better understanding of these processes will allow us to select specific TFs for use in genetic engineering and genome editing to improve the agronomic properties of grapevine